Figure 1.
Expression of Msi2 mRNA and protein in ESC.
(A) Primers for RT-qPCR were designed to be specific for both isoforms of Msi2, isoform 1 or isoform 2. Primer set F1/R1 amplifies sequences present in both isoform 1 and 2. Primer sets F2/R2 and F3/R3 are specific to isoform 1 and isoform 2, respectively. (B) DNA fragments generated via RT-qPCR using RNA isolated from D3 ESC and the primer sets indicated were separated over a 2% agarose gel. (C) Western blot analysis of Msi2 isoforms expressed in nuclear extracts isolated from D3 ESC. Isoform 2 runs slightly faster than isoform 1 (35.7 kDa and 36.9 kDa, respectively) [5]. HDAC1 protein was used as a loading control.
Figure 2.
Knockdown of Msi2 results in the differentiation of ESC.
(A) Regions of Msi2 mRNA targeted by shRNA #1, shRNA #4, and shRNA #5. (B) The D3 ESC were infected with lentiviruses that express scrambled (Scr) shRNA, shRNA #1, shRNA #4, or shRNA #5 sequences. Two days after infection, the cells were subjected to puromycin selection for 24 hours. After selection, the cells were subcultured and grown for an additional 24 hours before nuclear extracts were harvested for western blot analysis. HDAC1 was used as the loading control for quantification. (C) Bright field photomicrographs of cells subcultured at 5,000 cells per cm2 were taken 7 days post-infection with each shRNA (left columns). Cells were stained with alkaline phosphatase (right column) 10 days post-infection after being subcultured at 200 cells per cm2. Arrows point to colonies that exhibit a morphology characteristic of ESC (ESC), a morphology consisting of ESC and differentiated cells (Mixed), or a morphology characteristic of differentiated cells (Diff).
Figure 3.
Knockdown of Msi2 decreases the cloning efficiency of ESC.
D3 ESC infected with lentiviral constructs that express Scr, Msi2#1, Msi2#4, or Msi2#5 shRNA sequences were subcultured at 200 cells per cm2. Six days later an observer unaware of sample designation scored colonies as ESC, Mixed or differentiated (Diff).
Table 1.
Genes more highly expressed (>2.5-fold) in Msi2-shRNA #1 treated ESC.
Table 2.
Genes more highly expressed (>2.5-fold) in control scr-shRNA treated ESC.
Figure 4.
Knockdown of Msi2 in ESC leads to the expression of lineage-specific markers.
Seven days post-infection, RNA was isolated from the D3 ESC infected with lentiviruses that express either the scrambled (Scr) shRNA sequence or the Msi2 shRNA #1 sequence. Microarray analysis was used to assess global changes in gene expression (Fig. S2A). RNA expression for a select set of genes (yellow bars) is indicated as fold change, where a value >1 represents an increase in expression in cells treated with shRNA #1. All microarray data is available on Gene Expression Omnibus (Accession No. GSE33882, GEO, http://www.ncbi.nlm.nih.gov/geo/). Expression of this gene subset was verified by RT-qPCR (black bars). Threshold cycle (Ct) values were calculated by normalizing all Ct values to GAPDH then subtracting the Ct value for cells infected with Msi2 shRNA #1 from the Ct value for cells infected with the scrambled shRNA lentivirus. A positive Ct value indicates an increase in the level of the transcript in the Msi2 knockdown cells. Multiple rounds of RT-qPCR were used to calculate an average change in Ct value, and error bars represent standard deviation.
Figure 5.
Overexpression of Msi2 in ESC increases the cloning efficiency of ESC.
(A) Western blot analysis of Msi2 present in D3 ESC stably infected with a lentivirus for the inducible expression of Flag-tagged Msi2 isoform 1 (left) or Flag-tagged Msi2 isoform 2 (right) in the presence of 1 µg/ml Dox. (B) Effects of inducing Flag-tagged Msi2 isoform 1 (left) or Flag-tagged Msi2 isoform 2 (right) on the cloning efficiency of D3 ESC. The cells were plated at clonal density (200 cells per cm2) and exposed to 1 µg/ml Dox for 5 days. The error bars are standard error of the mean. This experiment was repeated twice and similar results were obtained.
Figure 6.
Rescue of Msi2 knockdown using an epitope-tagged overexpression construct.
Cells engineered to overexpress Flag-tagged Msi2 isoform 1 or Flag-tagged Msi2 isoform 2, i-Msi2.1-D3 and i-Msi2.2-D3 respectively, were plated at 10,500 cells/cm2 in the presence or absence of 2 µg/ml Dox. Cells were infected with lentiviral constructs that express Msi2 shRNA #1 for 48 hours. One day later, the cells were subcultured at 600 cells/cm2 in the continued presence or absence of 2 µg/ml Dox and the remaining cells harvested for protein extracts. Western blot analysis was used to monitor the levels of Msi2 in i-Msi2.1-D3 (A) or i-Msi2.2-D3 (B) stably infected with shRNA #1 lentivirus in the presence or absence of 2 µg/ml Dox. D3 ESC infected with the lentiviral vector that expresses the scrambled shRNA was used as a control. (C) Effects of inducing Flag-tagged Msi2 isoform 1 (left) or Flag-tagged Msi2 isoform 2 (right) on the cloning efficiency of D3 ESC following Msi2 knockdown. Six days following subculture, two observers unaware of sample designation scored colonies as ESC, Mixed or differentiated (Diff). The error bars are standard deviation between the average percentages as scored by the two observers. This experiment was repeated twice and similar results were obtained.
Figure 7.
Effects of Msi2 overexpression on the differentiation of ESC.
i-Msi2.1-D3 and i-Msi2.2-D3 were treated with or without 1 µg/ml Dox for 48 hours prior to plating at 4,500 cells/cm2. Cells were treated with and without Dox in the presence of 5 µM RA for 48 hours, followed by the removal of LIF for 48 hours to further the differentiation of the cells. RNA was isolated from these cells and the expression of a subset of differentially expressed genes was verified by RT-qPCR, as described in the Materials and Methods. Threshold cycle (Ct) values were normalized to GAPDH and represent the difference in transcript levels between the indicated treatment condition and undifferentiatied ESC. A positive Ct value indicates an increase in the level of the transcript in the differentiated cells, as indicated. Multiple rounds of RT-qPCR were used to calculate an average change in Ct value, and error bars represent standard deviation.
Table 3.
Oligonucleotides.